Recombinases, integrases and resolvases (collectively referred to herein as recombinases) mediate the site-specific recombination of DNA. These recombinases were first identified in phage that integrate into host chromosomes. Such integration allows the phage to remain latent in the cell as a prophage.
Site-specific recombinases catalyze conservative DNA rearrangements at specific target sequences. The 38 kDa Cre recombinase (cyclization recombination), derived from the bacteriophage P1, is a well characterized and widely used enzyme of the Integrase family (reviewed by Sauer, Methods, 14:381-392 (1998)). Cre plays two essential roles in the life cycle of P1: First, it provides a host-independent mechanism for P1's genome cyclization after infection, which can be important when the recombination system of the host is compromised. Second, Cre resolves dimerized P1 prophage plasmids to guarantee proper segregation during cell division.
Cre acts on a 34 bp sequence located on both ends of the linear P1 genome, that is called loxP (locus of crossover of P1; Sternberg and Hamilton, J. Mol. Biol., 150:467-486 (1981)), loxP consists of two 13 bp inverted repeats flanking a non-palindromic 8 bp core that defines the assigned direction of the sequence (as shown on the upper part of FIG. 1). Depending on this direction recombination catalyzed by Cre leads to excision of insertion of DNA flanked by loxP sites oriented in the same direction (indicated by loxP2), but leads to inversion when oriented in the opposite direction (FIG. 2).
In general, Cre-recombination involves the following four events: (i) DNA binding, (ii) synapsis (as defined below), (iii) cleavage, and (iv) strand exchange. To study this process in greater detail, mutants defective for each step have been isolated using several screening procedures (Wierzbicki et al., J. Mol. Biol., 195:785-794 (1987)). In addition, the crystal structure of Cre complexed with an artificial suicide substrate has been recently resolved, providing additional insights into site-specific recombination (Guo et al., Nature, 389:40-46 (1997)). From these studies, the following has been proposed: Four interacting Cre molecules are necessary for recombination between two lox sites, with each enzyme binding one inverted repeat plus the two outermost bp of the non-symmetric core region (DNA binding). This leads to the formation of a clamp, allowing DNA contacts in the major, as well as in the minor groove. In the step referred to as synapsis, the two lox sites with the bound Cre molecules, are aligned in parallel leading to an approximate 100° bending of the DNA. In the following step of strand cleavage, one of the two Cre molecules on each lox site causes a staggered cut in the core region, as indicated by the vertical arrows in FIG. 1. This leads to a 6 bp 5′ overhang and a covalent 3′ phosphotyrosine linkage between the catalytic residue tyrosine 324 of Cre and the guanine (position 4) at the cleaving site of loxP. The created phosphotyrosine intermediate is thought to provide the energy for the reaction, thereby explaining why Cre does not require an external energy source. In the next step, the first strand is exchanged between the two nicked lox sites, creating an intermediate, named Holliday structure (Sigal and Alberts, J. Mol. Biol., 71:789-793 (1972)). Of note, this first strand exchange is asymmetric, since the bottom strand (FIG. 1) is always exchanged first (Hoess et al., Proc. Natl. Acad. Sci. USA, 84:6840-6844 (1987)). During the final step, the second strand is exchanged and Cre released from its substrate.
Because of the simplicity and the ability of Cre to function in yeast and mammalian cells (Sauer, B., Mol. Cell. Biol., 7:2087-2096 (1987); Sauer and Henderson, Proc. Natl. Acad. Sci. USA, 85:5166-5170 (1988), Sauer and Henderson, Nucl. Acids Res., 17:147-161 (1989), and Sauer and Henderson, The New Biologist, 2:441-449 (1990), Cre assisted site-specific recombination has become an important tool for efficient, specific, and conditional manipulations of eukaryotic genomes (Lakso et al., Proc. Natl Acad. Sci. USA, 89:6232-6236 (1992)): Kilby et al., Genet., 9:413-421 (1993); Sauer, B., Meth. enzymol., 225:890-900 (1993); Kühn et al., Science, 269:1427-1429 (1995); Metzger et al., Pro. Natl. Acad. Sci. USA, 92:6691-6995 (1995).
However, there are some inconveniences for the successful use of Cre-related technologies, that include the following: (i) lox sites need to be introduced by homologous recombination at the desired region into the genome before Cre can be used, (ii) the frequency of correct site-specific recombination due to Cre expression is not 100%, and consequently, (iii) selectable markers are necessary in most strategies involving Cre for genome manipulation in higher eukaryotes. These markers, e.g. neo or TK, may introduce problems in subsequent studies, particular in those related to animal development. The number of available selectable markers that can be used in limited also. Additional site-specific recombinases that also function efficiently in eukaryotic systems, but recognize different sites from lox would be helpful. Similar inconveniences limit the usefulness of other recombinases.
Therefore, it is an object of the present invention to provide method of identifying variant recombinases that can mediate recombination between variant recombination sites.
It is another object of the present invention to provide variant recombinases that can mediate recombination between variant recombination sites.
It is another object of the present invention to provide a method of recombining nucleic acid molecules in vitro and in vivo.
It is another object of the present invention to provide Cre variants that recognize variant recombination sites.